The document outlines the principles and protocols for toxicology testing, particularly the OECD test guidelines for a 28-day repeated dose toxicity study in rodents, focusing on safety assessment for potential drugs. It discusses various toxicity testing types, animal care conditions, dosages, and monitoring practices to identify adverse effects, including neurotoxic and endocrine disruptor evaluations. Key parameters for data collection and analysis for hazard identification and risk assessment are also summarized.

2.  Toxicology is the branch of science that deals with toxins and
poisons, their effects and treatment.
 Toxicological screening is very important for development of
new drugs
 Significance:
It helps to calculate the no observed adverse effects
level(NOAEL)dose and it is helpful for clinical trials.

3.  General Toxicity testing :
Is referred to as series of toxicity testing required by
International regulatory for
compliance to Good Laboratory Practices (GLP) for proof of
safety in experimental animal prior to their testing in
human.

4.  It comprises of : Acute, Sub-acute and chronic toxicity.
 These studies are conducted based on Guidelines given by
the regulatory agencies,
ICH
FDA
OECD
WHO
EPA
Schedule Y

5.  The effect of exposure is generally for 28 days.
 OECD Test Guidelines describing short‐term repeat‐dose
toxicity test item administered via different routes are:
 Goal: To estimate safety margin of test item
ROUTE TG(test guideline)
Oral 407
Dermal 410
Inhalation 412

6.  The original OECD Test Guideline 407 was adopted in 1981.
 In 1995 a revised version was adopted, to obtain additional
information from the animal used in the study, in particular
on neurotoxicity and immunotoxicity.
 Amended in 1998, to obtain information related to endocrine
disruptors.

7. Principle of the Test
OECD 407
Test substance
administered once daily,
by oral route, for 28 d.
Animals observed closely,
each day for signs of
toxicity
OECD 410
Test substance
administered dermally for
28 days. Observed
closely, each day for signs
of toxicity
OECD 412
Test substance -
inhalation administered
for a defined period.
Observed closely, each
day for signs of toxicity
Allows to determination of the No-Observed Adverse Effect
Level (NOAEL) and provide information on selection of
doses for long term studies

9.  Determination of oral toxicity using repeated doses may be
carried out after initial information on toxicity has been
obtained by acute toxicity testing.
 This TG is intended to investigate effects on the nervous,
immune and endocrine systems.
 It should identify chemicals with neurotoxic potential and
chemicals that interfere with thyroid physiology.
 It may also provide data on chemicals that affect the male and
or female reproductive organs in young adult animals and may
give an indication of immunological effects.

10.  The results from the TG 407 should be used for hazard
identification and risk assessment. The results obtained by
the endocrine related parameters should be seen in the
context of the “OECD Conceptual Framework for Testing and
Assessment of Endocrine Disrupting Chemicals”.
 The international program conducted on the validation of
parameters suitable to potentially detect endocrine activity of
test substance.
 A variety of parameters were found to be indicative of
endocrine-related toxicity and have been incorporated in the
TG. Parameters for which insufficient data were available to
prove usefulness or which showed only weak evidence in the
validation programme of their ability to help in detection of
endocrine disrupters are proposed as optional endpoints.

11. Mandatory
endpoints
Optional endpoints
Weight
Testes Ovaries
Epididymides Uterus, Including
cervix
Adrenals Thyroid
Prostate +
seminal
vesicles
Histopathology
Gonads Vaginal smears
-Testes Male mammary
glands
-Ovaries Pituitory
Accessory sex organs :
-Epididymides,
-Prostate + seminal vesicles
Uterus, including cervix
-adrenal
-thyroid
-vagina
Hormonal Measurements
T3 and T4
TSH

12.  On the basis of data generated in the validation process, it
must be emphasized that the sensitivity of this assay is not
sufficient to identify all substances with (anti)androgenic or
(anti)oestrogenic modes of action.
 The TG nevertheless, during the validation process identified
compounds weakly and strongly affecting thyroid function,
and strong and moderate endocrine active substances acting
through oestrogen or androgen receptors, but in most cases
failed to identify endocrine active substances that weakly
affect oestrogen or androgen receptors. Thus it can’t be
described as a screening assay for endocrine activity.

13.  The test substance is orally administered daily in graduated doses to
several groups of experimental animals, one dose level per group for a
period of 28 days.
 During the period of administration the animals are observed closely, each
day for signs of toxicity.
 At the end of the study, the experimental animals are sacrificed. Gross
Pathological changes are observed and all the tissues are subjected to
histopathological analyses.
 The data obtained by this TG Allows to determination of the No-
Observed Adverse Effect Level (NOAEL) and provide information on
selection of doses for long term studies

14. Subacute (Guidelines 407)
SD Rats, male and Female
Control Low dose
Medium
dose High dose

16.  The preferred rodent species is the rat, although other rodent
species may be used (Young healthy adult animals should be
employed)
 If the parameters specified within this TG 407 are investigated
in another rodent species a detailed justification should be
given.
 Other species should respond to toxicants in a similar manner
to the rat.

17.  The use of smaller species may result in increased variability due
to technical challenges of dissecting smaller organs.
 In the international validation program for the detection of
endocrine disrupters, the rat was the only species used.
 Female rats should be nulliparous and non pregnant.
 When a repeated oral dose is conducted as a preliminary to a longer-
term study, it is preferable that animals from the same strain and
source should be used in both studies.

18.  Dosing should begin as soon as feasible after weaning, and,
in any case, before the animals are nine weeks old.
 weight variation of animals used should be minimal and
not exceed ± 20%

19.  Husbandry conditions :
Temperature : 22°C (± 3°C)
RH : 30%- 70%
Lighting should be artificial, 12 hours light, 12 hours dark.
 Feeding:
• Conventional laboratory diets may be used with an unlimited supply of
drinking water.
• For group caging, not more than five animals should be housed per cage.
• The feed should be regularly analysed for contaminants.
• A sample of the diet should be retained until finalisation of the report.

20.  Healthy young adult animals grouped in test and control.
 Cages should be arranged in such a way that possible
effects due to cage placement are minimized.
 The animals are identified uniquely.
 Kept in their cages for at least five days prior to the start of
the treatment study to allow for acclimatisation to the
laboratory conditions.

21.  The test compound is administered by gavage or via the diet
or drinking water.
 Test substance is dissolved or suspended in a suitable
vehicle.
Vehicles examples
Water
Methylcellulose or carboxymethylcellulose
Oil(corn, peanut, sesame)

22. At least 10 animals (five female and five male) in each
group
 If interim euthanasia are planned, the number should be
increased by the number of animals scheduled to be
euthanised before the completion of the study.
 Satellite group: in the control and in the top dose group
for observation of reversibility, persistence, or delayed
occurrence of toxic effects, for at least 14 days post
treatment.

23. 60 animals
30males+30 females
Control
(5M+5F)
Control satellite
group
Low dose
(5M+5F)
Medium
dose
(5M+5F)
High dose
(5M+5F)
High(top)dose satellite
group
6 groups
total

24.  Generally, at least three test groups and a control group
should be used, but if from assessment of other data, no
effects would be expected at a dose of 1000mg/kg, a limit
test may be performed.
1000 mg/kg no toxicity effect
Toxicity effect No need for subacute toxicity
studies
Subacute toxicity studies

25.  If there are no suitable data available, a range finding study
(animals of the same strain and source) may be performed to
aid the determination of the doses to be used.
 Control group should receive the vehicle.

26. First High dose is
decided
Low Dose.
- Should not produce
any observed toxic
effect
- May be 10 times
lower than the
medium dose levels
or higher dose levels
Medium Dose
Usually 2 to 4 times
reduced as
compared to the
highest dose levels.
High Dose
This dose should
induce toxic effect
but not severe
toxicity or death

27.  Dose levels should be selected taking into account
any existing toxicity and (toxico-) kinetic data
available for the test compound or related materials.
 Two to four fold intervals are frequently optimal for
setting the descending dose levels and addition of a
fourth test group is often preferable to using very
large intervals Intervals (e.g. more than a factor of
10) between dosages.

28.  The animals are dosed with test substance daily 7 days each week for
a period of 28 days.
 When the test substance is administered by gavage, this should be
done in a single dose
 The volume should not exceed 1 ml/100g body weight except in the
case of aqueous solutions where 2 ml/100 g body weight may be
used.
 Ensure a constant volume at all dose levels.
 Substances administered via the diet or drinking water it is important
to ensure that test substance do not interfere with normal nutrition or
water balance.
 Where a repeated dose study is used as a preliminary to a long term
study, a similar diet should be used in both studies.

29.  The observation period should be 28 days
 Animals in a satellite group observed for 14
days without treatment to detect delayed
occurrence, or persistence of, or recovery from
toxic effects.
 Observations should be made at least once a
day, preferably at the same time.
 At least twice daily, all animals are observed
for morbidity and mortality.

30.  Before the first exposure : Detailed clinical observations
should be made in all animals.
 These observations should be made outside the home cage
in a standard arena and preferably at the same time of day
on each occasion.
 They should be carefully recorded, preferably using scoring
systems, explicitly defined by the testing laboratory.
 Signs noted should include,
 Changes in skin, fur, eyes, mucous membranes.
 Autonomic activity (e.g. lacrimation, piloerection, pupil size,
unusual respiratory pattern).

31.  In the fourth exposure week:(different types of stimuli
are observed e.g. auditory and visual )
 Assessment of grip strength and motor activity
 At necropsy, the oestrus cycle of all females could be
determined (optional).
 In the presence of observed general toxicity (e.g.
reduced body weight, liver , heart, lung or kidney
effects, etc.) or other changes that may not be toxic
responses (e.g. reduced food intake, liver enlargement)
 Observed effects on immune, neurological or endocrine
sensitive endpoints should be interpreted with caution.

32.  Body weight/body weight changes.
• All animals should be weighed at least once a week.
 Food/water consumption.
• Measurements of food consumption should be made at
least weekly.
• If the test substance is administered via the drinking
water, water consumption should also be measured at
least weekly.

33. Haematology :Blood samples should be collected just prior
to euthanasia of the animals and stored under appropriate
conditions.
The following haematological examinations :
1. Haematocrit,
2. Haemoglobin concentrations,
3. Erythrocyte count and Reticulocytes,
4. Total and differential leucocyte count, platelet count and
a measure of blood clotting time.

34. Rat
RBC(x10v /mm³) 7-10
PCV(%) 36-18
Hb(g/dl) 11-18
WBC(X10³/m³) 6-17
Neutrophils(%) 9-34
Lymphocytes(%) 65-85
Eosinophils(%) 0-6
Monocytes(%) 0-5
Basophils(%) 0-1.5
Platelets(X10³/mm³) 500-1300

35.  Investigations of plasma or serum shall include sodium,
potassium, glucose, total cholesterol, urea, creatinine, total
protein and albumin.
 Optionally, the following urinalysis determinations could be
performed during the last week of the study using timed urine
volume collection;
Appearance, volume, osmolality, pH, protein, glucose and
blood/blood cells.

36. Rat
Protein (g/dl) 5.6-7.6
Albumin (g/dl) 2.8-4,8
Globulin (g/dl) 1.8-3
Glucose (mg/dl) 50-135
Urea nitrogen 15-21
Creatinine (mg/dl) 0.2-0.8
Bilirubin (mg/dl) 0.2-0.55
Cholesterol (mg/dl) 40-130

37.  RESPIRATORY :
Blockage in the nostrils, changes in rate & depth of
breathing, changes in color of body surfaces
 Signs like dyspnea, abdominal breathing, Apnea.

38.  Changes in frequency & nature of movements
 Signs like decrease/increase in spontaneous motor activity,
 Anesthesia
 Ataxia
 Tremors

39.  Lacrimation, Chromodacryorrhea ( red lacrimation )
 Miosis and Mydriasis.

40.  Bradycardia, tachycardia,
 Arrhythmias.
 All signs indicates involvements of CNS, Sensory, autonomic and
neuromuscular systems.

41.  Salivation
 Piloerection
 Analgesia
 Muscle tone - hypotonia - hypertonia
 GIT signs: Emesis

42.  Although in the international evaluation of the endocrine
related endpoints a clear advantage for the determination of
thyroid hormones (T3, T4) .
 It may be helpful to retain plasma or serum samples to
measure T3, T4 and TSH (optional) if there is an indication
for an effect on the pituitary-thyroid axis.
 These samples may be frozen at -20° for storage.
 Plasma samples specifically intended for hormone
determination should be obtained at a comparable time of the
day.
 The numerical values obtained when analysing hormone
concentrations differ with various commercial assay kits.

43.  All animals in the study shall be subjected to a full, detailed
gross necropsy which includes careful examination of the
external surface of the body, all orifices, and the cranial,
thoracic and abdominal cavities and their contents.
 The liver, kidneys, adrenals, testes, epididymides, prostate +
seminal vesicles ,thymus, spleen, brain and heart of all
animals (apart from those found moribund and/or euthanised
prior to the termination of the study)
 In addition, two other tissues could be optionally weighed as
soon as possible after dissection, to avoid drying: paired
ovaries (wet weight) and uterus, including cervix .
 The thyroid weight (optional) could be determined after
fixation.

44.  Full histopathology should be carried out on the preserved
organs and tissues of all animals in the control and high dose
groups.

45.  Data should be summarised in tabular form.
 All the possible, numerical results should be evaluated by an
appropriate and generally acceptable statistical method.

46. 14-28 days
Group Rodents Non Rodents
M F M F
Low dose 6-10 6-10 2-3 2-3
Moderate
dose
6-10 6-10 2-3 2-3
High dose 6-10 6-10 2-3 2-3
 Numbers of animals required for Repeated dose toxicity studies

47.  OECD Guideline for the Testing of Chemicals, 407
Adopted 3 October 2008 Repeated Dose 28-day Oral
Toxicity Study in Rodents.
 CARE V. CPCSEA guidelines for laboratory animal facility.
Indian Journal of Pharmacology. 2003;35:257-74.

48. Thank you